Project Summary/Abstract The mounting of an antibody response depends on cells positioning within lymphoid organs at sites where they can encounter antigen and then relocalize to achieve specific cell-cell interactions and exchange signals necessary for lymphocyte differentiation. T follicular helper (Tfh) cells are a specialized T cell type that is critical for mounting most antibody responses yet the cues instructing development of these cells have not been fully elucidated. Very recently we have found that Epstein Barr Virus-Induced gene-2 (EBI2 or GPR183) guides activated helper T cells to the outer T zone where they interact with activated DCIR2-expressing dendritic cells (DCs). These interactions promote Tfh cell differentiation and this involves a novel mechanism where CD25 (IL2R?) produced by the DCs quenches T cell-derived IL2. The first Aim of this proposal will investigate how DC-T cell interactions in the outer T zone favor Tfh cell differentiation. This will include a detailed exploration of the conditions promoting CD25 expression and shedding by DCs and an assessment of how this soluble receptor controls IL2 availability. Two-photon microscopy will be used to visualize the impact of EBI2-deficiency on helper T cell ? DC interactions. The role of DC-derived CD25 in controlling additional T cell differentiation events, including Th17 cell induction, will be examined. As well as its role in B cells and helper T cells, EBI2 functions to promote the positioning and homeostasis of CD4+ DCIR2+ DCs. Until now, all EBI2 functions have been thought to be directed by the ligand 7?,25- dihydroxycholesterol (7?,25-HC). The second aim will follow-up on new evidence that CD4+ DCIR2+ DC positioning and homeostasis requires the enzyme Cyp27a1, likely acting to generate a second EBI2 ligand, 7?,27-HC. The Aim will measure 7?,27-HC levels, determine Cyp27a1 enzyme distribution and mechanism of action, and test the role of this pathway in humoral immunity. The mechanisms responsible for positioning of activated DCs in the outer T zone will also be explored. Mounting appropriately regulated immune responses is essential for human health. This work will define new cellular and molecular requirements for the earliest steps necessary for promoting appropriately tuned antibody responses to vaccine antigens and infectious agents. The research will also provide insight relevant to understanding factors that influence the therapeutic actions of IL2 and how CD25 expression by DCs influences tumor immune responses.